Recombinant non-human mammalian model for hepatitis infection and immunopathogenesis

ABSTRACT

Provided herein is a recombinant non-human mammal having an immune system including human immune cells and having a liver including human liver cells, and methods for producing the same. Also provided are methods of screening a compound for activity in treating hepatitis, comprising: administering a test compound to a recombinant non-human mammal as described herein; and then detecting the presence or absence of said activity in said mammal (e.g., by biochemical assay), said presence of said activity in said mammal indicating that said compound has activity in treating hepatitis. Methods of making fusion cells useful for the production of human monoclonal antibodies are also provided.

RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 12/918,676, filed Sep. 15, 2010, now U.S. Pat. No.9,173,383, which is a 35 U.S.C. §371 national phase entry of PCTApplication PCT/2009/001081, filed Feb. 20, 2009, and published inEnglish on Aug. 27, 2009, as International Publication No. WO2009/105244, and which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/030,328, filed Feb. 21, 2008, the disclosure ofeach of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under grants R21-CA99939and RO1-AI41356 from the National Institutes of Health. The UnitedStates Government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns transgenic non-human animals and methodsof making and using the same.

BACKGROUND OF THE INVENTION

Liver disease induced by hepatitis C virus (HCV) and hepatitis B virus(HBV) is a global health problem. The World Health Organizationestimates that 350-400 million people are chronically infected, andabout one million die annually due to chronic hepatitis, cirrhosis orliver cancer. Another 200 million people are infected by HCV, of whom70%-85% will become chronically infected. HBV and HCV infection is theleading cause of liver disease in Asia. In Western countries, HCVinfection is the leading indication for liver transplantation and amajor cause of liver cancer.

The liver is a unique organ for immune responses and viruses/tumors (forreview, see Crispe, I. N. 2003. Hepatic T cells and liver tolerance. NatRev Immunol 3:51-62). The liver intrinsically dampens the immuneresponses to foreign antigens filtering through it from the intestines.An allogeneic liver transplant is often accepted with minimal or noimmune suppression. Tumors with tumor-specific antigens can metastasizeto and survive in the liver in immuno-competent patients. Infection ofhepatocytes by viruses such as HCV often leads to specific immunetolerance to the virus, and to chronic or persistent infection (Bowen etal. 2005. Adaptive immune responses in acute and chronic hepatitis Cvirus infection. Nature 436:946-52; Grakoui et al. 2003. HCV persistenceand immune evasion in the absence of memory T cell help. Science302:659-62).

Although the liver may provide an immunologically privileged site forinfections, most infections in the liver, such as HAY and MHV, and HBVin adults, are effectively cleared and accompanied with lastingprotective immunity. It is unusual that greater than 80% of HCVinfection in immuno-competent hosts leads to persistent infection.HCV-encoded factors and/or unique host cell tropism may contribute tothe efficient immune evasion and HCV persistence.

The liver consists of unique subsets of antigen presenting cells andlymphocytes. In addition to dendritic cells, a large number of livermacrophages (or Kupffer cells) and sinusoidal endothelial cells in theliver also have efficient phagocytosis activity and express variouslevels of MHC and T cell costimulatory molecules. However, they oftenshow suboptimal T cell activation activity in vivo (Everett et al. 2003.Kupffer cells: another player in liver tolerance induction. LiverTranspl 9:498-9; Parker et al. 2005. Liver immunobiology. Toxicol Pathol33:52-62; Racanelli et al. 2006. The liver as an immunological organ.Hepatology 43:S54-S62; Sun et al. 2003. Hepatic allograft-derivedKupffer cells regulate T cell response in rats. Liver Transpl 9:489-97;Wiegard et al. 2005. Murine liver antigen presenting cells controlsuppressor activity of CD4+CD25+ regulatory T cells. Hepatology42:193-9). The liver also contains lymphoid cells with unique features.Up to 25% of lymphoid cells belong to the NKT cell population thatexpresses TCR as well as NK markers. Their function in the liver is notclear, but they have been implicated in clearing infections in the liver(Behar et al. 1999. Susceptibility of mice deficient in CD1D or TAP1 toinfection with Mycobacterium tuberculosis. J Exp Med 189:1973-80; Skoldet al. 2003. Role of CD1d-restricted NKT cells in microbial immunity.Infect Immun 71:5447-55).

HCV/HBV coinfection with the HIV-1 virus, which is highly prevalentamong intravenous drug users, leads to accelerated liver diseaseprogression (Bani-Sadr et al. 2006. Hepatic steatosis in HIV-HCVcoinfected patients: analysis of risk factors. Aids 20:525-31; Brau, N.2003. Update on chronic hepatitis C in HIV/HCV-coinfected patients:viral interactions and therapy. Aids 17:2279-90; Sabin et al. 2004.HIV/HCV coinfection, HAART, and liver-related mortality. Lancet364:757-8; author reply 758). Liver failure is increasingly affectingHIV-1/HCV-coinfected patients, as their AIDS-free survival is beingprolonged by highly active antiretroviral therapy (HAART).

The available treatment for HCV infection is far from optimal, andHIV-1/HCV-coinfected patients show even worse responses to pegylatedinterferon plus rivabirin than HCV-monoinfected patients (Sola et al.2006. Poor response to hepatitis C virus (HCV) therapy in HIV- andHCV-coinfected patients is not due to lower adherence to treatment. AIDSRes Hum Retroviruses 22:393-400). There is a great need for alternativetreatment options for hepatitis infection.

A relevant small animal model for research on HCV/HBV infection andpathogenesis is therefore needed. However, HCV fails to infect murinecells due to blocks at multiple steps of the HCV life cycle. HCV and HBVcan only infect, establish chronic infection and to lead to liverpathogenesis in humans. Only a reduced chronic infection andimmuno-pathogenesis are observed in chimpanzees, which provides the onlycurrent non-human animal model for HCV infection (Pietschmann et al.2003. Tissue culture and animal models for hepatitis C virus. Clin LiverDis 7:23-43).

The Alb-uPA transgenic mouse, developed in 1990 by Heckel et al. (1990.Neonatal bleeding in transgenic mice expressing urokinase-typeplasminogen activator. Cell 62:447-56) to study plasminogenhyperactivation and therapeutic protocols to prevent bleeding, containsa tandem repeat of four murine uPA genes under the control of an albuminpromoter. The transgene overexpression results in profoundhypo-fibrinogenemia and accelerated hepatocyte death. Homozygous animalscan be rescued by transplantation of murine or human hepatocytes, whichundergo rapid proliferation to replace the dying hepatocytes (Mercer etal. 2001. Hepatitis C virus replication in mice with chimeric humanlivers. Nat Med 7:927-33; Meuleman et al. 2005. Morphological andbiochemical characterization of a human liver in a uPA-SCID mousechimera. Hepatology 41:847-56; Meuleman et al. 2006. Immune suppressionuncovers endogenous cytopathic effects of the hepatitis B virus. J Virol80:2797-807). Transplanted human hepatocytes can be infected with HBVand HCV (Mercer et al. 2001. Hepatitis C virus replication in mice withchimeric human livers. Nat Med 7:927-33; Meuleman et al. 2006. Immunesuppression uncovers endogenous cytopathic effects of the hepatitis Bvirus. J Virol 80:2797-807).

A molecularly cloned, cell culture-produced hepatitis C virus (HCVcc)genome has been recently shown to support efficient replication in vitro(Blight et al. 2000. Efficient initiation of HCV RNA replication in cellculture. Science 290:1972-4; Lindenbach et al. 2005. Completereplication of hepatitis C virus in cell culture. Science 309:623-6) andin vivo (Lindenbach et al. 2006. Cell culture-grown hepatitis C virus isinfectious in vivo and can be recultured in vitro. Proc Natl Acad SciUSA 103:3805-9). The HCVcc is infectious in uPA-SCID mice reconstitutedwith human hepatocytes, and infection can be serially passaged to anaïve animal.

Infectivity of HCV can be studied in the uPA-SCID mice transplanted withhuman hepatocytes (Kneteman et al. 2006. Anti-HCV therapies in chimericscid-Alb/uPA mice parallel outcomes in human clinical application.Hepatology 43:1346-53; Lindenbach et al. 2005. Complete replication ofhepatitis C virus in cell culture. Science 309:623-6; Mercer et al.2001. Hepatitis C virus replication in mice with chimeric human livers.Nat Med 7:927-33; Meuleman et al. 2005. Morphological and biochemicalcharacterization of a human liver in a uPA-SCID mouse chimera.Hepatology 41:847-56). However, immuno-pathogenesis cannot because uPAmice have no immune system. In addition, the uPA-SCID mouse is very sickand not suitable for many studies.

The RagFahγC TKO mouse also allows efficient engraftment of humanhepatocytes in a uPA transgene-dependent fashion (Azuma et al. 2007.Robust expansion of human hepatocytes in Fah(−/−)/Rag2(−/−)/I12rg(−/−)mice. Nat Biotechnol 25:903-10). In the B6 Rag/γC DKO background, thefumarylacetoacetate hydrolase (Fah) mutation is crossed to generate theRagFahγC triple KO mice. After pretreatment with a urokinase-expressingadenovirus, these animals could be highly engrafted with humanhepatocytes. However, due to lack of a functional immune system (whichis not suitable for human immune system development), it is not possibleto study HCV/HBV immunopathogenesis in these uPA-SCID/-TKO models.

Thus, a mouse model having both a functional human immune system and ahuman liver is needed to study HCV/HBV infection, immune responses andpathogenesis.

Two human-mouse chimera models with human lymphoid organs implanted inimmunodeficiency mice have been constructed to study HIV-1 infection invivo. The hu-PBL-SCID mouse is limited due to its lack of humanhemato-lymphoid organs and its selective engraftment of xeno-reactivehuman T cells (Mosier et al. 1988. Transfer of a functional human immunesystem to mice with severe combined immunodeficiency. Nature 335:256-9;Mosier et al. 1991. Human immunodeficiency virus infection ofhuman-PBL-SCID mice. Science 251:791-4; Tary-Lehmann et al. 1994.Anti-SCID mouse reactivity shapes the human CD4+ T cell repertoire inhu-PBL-SCID chimeras. J Exp Med 180:1817-27). The SCID-hu Thy/Liv mousehas an intact human thymus organ, which allows investigation of HIV-1pathogenesis in the thymus (McCune et al. 1991. The SCID-hu mouse: asmall animal model for HIV infection and pathogenesis. Annu Rev Immunol9:399-429; McCune et al. 1988. The SCID-hu mouse: murine model for theanalysis of human hematolymphoid differentiation and function. Science241:1632-9; Su et al. 1995. HIV-1-induced thymocyte depletion isassociated with indirect cytopathogenicity and infection of progenitorcells in vivo. Immunity 2:25-36). However, no human B or myeloid cellsand very low levels of human T cells are detected in the peripheralorgans or blood. Therefore, no significant primary human immuneresponses are observed in the model.

A more relevant in vivo non-human animal model that allows hepatitisinfection as well as hepatitis and HIV co-infection is, therefore,needed.

SUMMARY OF THE INVENTION

Provided herein is a recombinant non-human mammal (e.g., a mouse)comprising: (a) an immune system comprising, consisting of, orconsisting essentially of: human T cells, human B cells, human naturalkiller cells, human monocytes and macrophages and human dendritic cells,so that the mammal expresses a human immune system phenotype; and (b) aliver comprising, consisting of, or consisting essentially of humanhepatocytes, so that the mammal expresses a human liver phenotype. Insome embodiments, the human liver cells comprise at least 20, 30, 40 or50% of said liver of said mammal (by weight, by volume and/or by numberof cells) (measured, e.g., by human albumin staining). In someembodiments, the mammal is a Rag2-gammaC double knockout mammal. In someembodiments, the mammal comprises non-human cells that contain aliver-specific promoter (e.g., an albumin promoter) operativelyassociated with a nucleic acid encoding a product with inducibletoxicity to said non-human cells (e.g., FKBP-Caspase 8). In someembodiments, the mammal is infected with a virus, e.g., HIV-1 virus, ahepatitis virus (e.g., HBV or HCV), or both.

Also provided herein are methods of screening a compound for activity intreating hepatitis, comprising: administering a test compound to arecombinant non-human mammal as described herein; and then detecting thepresence or absence of said activity in said mammal (e.g., bybiochemical assay), said presence of said activity in said mammalindicating that said compound has activity in treating hepatitis.

Further provided are methods of making a non-human transgenic mammalcomprising an immune system, said immune system comprising: human Tcells, human B cells, human natural killer cells, human monocytes andmacrophages and human dendritic cells, so that the mammal expresses ahuman immune system phenotype; and a liver comprising human hepatocytes,so that the mammal expresses a human liver phenotype. The methodscomprise the steps of: a) providing a BalbC/Rag2^(−/−)γ_(c) ^(−/−)double knockout transgenic mammal (optionally further comprising anAlb-FKBP-Casp8 transgene); b) transplanting human CD34+ hematopoieticstem cells into said double knockout transgenic mammal, wherein saidstem cells differentiate into human T cells, human B cells, humannatural killer cells, and human dendritic cells in said transgenicmammal; and c) transplanting human liver cells into said double knockouttransgenic mammal, wherein said liver cells form human hepatocytes insaid liver of said transgenic mammal. In some embodiments, the humanCD34+ hematopoietic stem cells and the human liver cells are autogeneic.In some embodiments, the human liver cells comprise human parenchymahepatoblasts. In some embodiments, the human CD34+ hematopoietic stemcells and the human liver cells are transplanted simultaneously. In someembodiments, the transplanting steps are carried out when the transgenicmammal is from 0 to 10 days old. In some embodiments, the methodsfurther comprise the step of administering (e.g., by injection) a c-Metagonist (e.g., an anti-C-met antibody) selective for human c-Met to saidtransgenic animal.

Methods of making fusion cells useful for the production of humanmonoclonal antibodies are also provided, said method comprising:isolating a human antibody-secreting B lymphocyte from a recombinantnon-human mammal (e.g., from a spleen or lymph node), said mammalcomprising: (a) an immune system comprising: human T cells, human Bcells, human natural killer cells, human monocytes and macrophages andhuman dendritic cells, so that said mammal expresses a human immunesystem phenotype; and (b) a liver comprising human hepatocytes, so thatsaid mammal expresses a human liver phenotype; and fusing saidantibody-secreting B lymphocyte with immortal cells (e.g., human ormouse myeloma cells) to form said fusion cells. In some embodiments, themammal is infected with a virus, e.g., HIV-1 virus, a hepatitis virus(e.g., HBV or HCV), or both.

Also provided is the use of a non-human transgenic mammal, cell or cellculture as described herein for the preparation of a composition ormedicament for carrying out a method of treatment as described herein,or for making an article of manufacture as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H. Long-term, stable human hemato-lymphopoiesis in HSC-DKOMice. Human fetal liver CD34+ cells were injected intra-hepatically innewborn (1-3 days old) DKO mice. At 12-50 weeks post transplant, thehuman CD45⁺, murine CD45⁻ cells in PBMC, lymph node (LN), spleen, bonemarrow and thymus were analyzed by multi-color FACS. FIG. 1A: Human CD45reconstitution (12-16 weeks old) in 5 cohorts of DKO-hu HSC mice from 5independent donor fetal livers. Shown is average % human CD45+ cells inPB of reconstituted DKO-hu HSC mice. Error bars indicate standard errorsof each cohort (n=numbers of mice/cohort). FIG. 1B: Human cellsreconstitution in PBMC of the transplanted mice. Individual barsrepresent percentage of human CD45+ cells of 12 mice from the samecohort (14 weeks post transplant). The darker inside bars indicate theportion of human CD3+CD4+ cells. FIG. 1C: Total number of splenocytesand thymocytes from a typical DKO-hu cohort (n=12) is compared to wildtype and DKO mice. Error bars are standard deviations. FIG. 1D: Stablereconstitution of naïve and resting human T cells. Human T cells from aDKO-hu mouse at 50 weeks are analyzed for expression of CD45RO and CD69.FIG. 1E/F: Human CD4+ T cells purified from DKO-hu mice or from humanPBMC are stimulated with various doses of anti-CD3 mAb with (solidsquares) or without (open squares) anti-CD28 mAb. Human T proliferationis measured by 3-H thymidine incorporation for 16 hr after 3 days postco-culture. FIG. 1G: Tolerization of human T cells to both BalbC hostcells and to the donor human cells in DKO-hu HSC mice. Splenocytes fromDKO-hu mouse cohort A (A1 and A2), cohort B (B) and untransplanted DKOmouse (DKO) are prepared and mixed in culture in triplicates at 2×10e5total cells/well (in mixed cultures, 1×10e5 cells per donor cells areused). Similar human cells (about 50%) are engrafted in A1, A2 and BDKO-hu mice. Only A1/B and A2/B co-culture show significantproliferation (p<0.05) in the MLR assay. FIG. 1H: DKO-hu mice arevaccinated with Ova protein and Ova-specific T cell recall response ismeasured at 3 weeks post vaccination. Splenocytes from vaccinated orcontrol “cohort-mate” mice are compared for response to Ova proteinchallenge in vitro. Human T cell proliferation is measured as above.

FIGS. 2A-2H. HIV-1 Replication and Pathogenesis in DKO-hu HSC mice.DKO-hu HSC mice were infected with HIV-R3A or JRCSF (5 ng p24/mouse).DKO mice or mock infected DKO-hu mice are used as controls. For R3Ainfection, plasma samples were collected at 1, 2, 3, 4 and 12 weeks postinfection and HIV genome copy numbers were determined with the RocheAmplicor HIV-1 Monitor Kit (FIG. 2A). (FIG. 2B) FACS analysis of humanCD45, CD3, CD4 and CD8 cells from blood samples are performed andrelative CD4 depletion is shown (as % CD3+CD4+ or CD4/CD8 ratio). Opensymbols are mock controls and solid symbols are HIV-infected mice. (FIG.2C/D) JRCSF infection is similarly analyzed at 1, 2, 4, 6, and 18 wpi.Shown are data from four infected DKO-hu mice and two mock control mice(FIG. 2D). (FIG. 2E-H) Spleen (FIG. 2E/F) or mLN (FIG. 2E/F) samplesfrom DKO-hu mice infected with R3A (FIG. 2E/G, R3A-2 wpi, 3 mocks and 4R3A-infected mice) or JRCSF (FIG. 2F/H, JRCSF-4 wpi, 4 mocks and 5JRCSF-infected mice) are summarized for relative CD4 and CD8 in humanleukocytes. Error bars are SE. *, p<0.05; **, p<0.01.

FIGS. 3A-3F. Reconstitution of human leukocytes (CD45+) and hepatocytes(Alb+). (FIG. 3A-B): Both T cells and myeloid cells were present in thereconstituted liver. Leukocytes from DKO-hu HSC/Hep mice were isolated.FIG. 3A: human CD45+CD3− cells were analyzed for CD4 and CD11cexpression. All myeloid cell types (monocytes and macrophage/Kupfercells; myeloid DC and PDC-CD123+, not shown) and B cells are present.FIG. 3B: human CD45+CD3+ cells were analyzed for CD4 and CD8 expression.(FIG. 3C-E) Liver sections from DKO-hu HSC/Hep mice at 5 weeks posttransplant were stained with anti-human albumin and DAPI. Human albumin+cells are detected in the liver parenchyma (FIG. 3C) or around thecentral vein (FIG. 3D). FIG. 3E: no primary antibody control. HumanAlbumin+ cells exist in the liver parenchyma. All slides arecounter-stained with DAPI for DNA (blue). FIG. 3F: When the humanalbumin levels in the blood were measured in a representative cohort(n=7), a steady level of human albumin (150-350 ng/ml) was detected from5-15 weeks post transplant. Pre-transplant sera were used as background(Non-tran).

FIGS. 4A-4C. Generation of the FKBP-Casp8 fusion gene with induciblecell killing activity. (FIG. 4A) Inducible activation of Caspase 8through dimerization. The chemical dimerizer AP20187 causesdimerization/activation of Caspase 8 through interaction of adjacentFKBP binding sites. M, myristoylation signal; FKBP, FK506 bindingdomain; caspase, human activated caspase 8 (fragment Ser217-Aps479. ref)was cloned into the pC4M-Fv2E vector (Ariad Pharmaceuticals) to expressthe FKBP2-Casp8 fusion protein whose activation is induced bydimerization with AP20187. (FIG. 4B-C) Dose-dependent induction ofapoptosis in cells transfected with FKBP-Caspase 8. 293T cells wereco-transfected with plasmids expressing eGFP alone, or with CMV-promoterdriven FKBP-Casp8. Transfected cells were cultured for 30 hours and thenvarious amounts of AP20187 dimerizer was added. The cells were thencultured for 24 hours, harvested, stained with 7AAD, and analyzed byFACS for GFP and 7AAD. (FIG. 4B) GFP+ (transfected) cells were gated andthe percentage of dead cells (7AAD+) was analyzed. (FIG. 4C)GFP-(untransfected bystander) cells were similarly analyzed. At least 3independent experiments are repeated.

FIGS. 5A-5C. Generation of Alb-FKBP-Casp8 transgenic DKO (AFC8/DKO)mice. (FIG. 5A) The Alb-FKBP-Casp8 transgene structure and AFC8/DKO-huHSC/Hep mice: the FKBP2Casp8 fusion gene was cloned into the liverspecific transgenic construct with the Albumin promoter (see Heckel etal. 1990. Neonatal bleeding in transgenic mice expressing urokinase-typeplasminogen activator. Cell 62:447-56). (FIG. 5B) Hepatocyte-specificapoptosis with expression from the albumin promoter/enhancer.Alb-FKBP-Casp8 functions in HepG2 but not 293T cells. As described inFIG. 5B, the FKBP-Casp8 gene controlled by CMV enhancer or thehepatocyte-specific albumin promoter was co-transfected withGFP-expressing plasmid in 293T and HepG2 cells. Transfected cells werecultured for 30 hours and AP20187 dimerizer (2 nM) was added to theculture medium. The cells were then cultured for 24 hours, harvested,stained with 7AAD, and analyzed for GFP and 7AAD. GFP+ cells wereanalyzed for 7AAD uptake (dead cells). (FIG. 5C) Generation ofAlb-FKBP-Casp8/DKO transgenic mice. Standard transgenic mouse procedurewas used to inject the transgene construct into fertilized DKQ embryos.In the initial screening of 11 mice from two injections, one transgenicfounder was identified. PCR is run using primers that amplify thetransgenic construct or the mouse endogenous p18 gene (see Kovalev etal. 2001. An important role of CDK inhibitor p18(INK4c) in modulatingantigen receptor-mediated T cell proliferation. J Immunol 167:3285-92).300 fg of transgene plasmid DNA, 100 ng of mouse genomic DNA, a mixtureof 300 fg plasmid+100 ng mouse DNA, Water+PCR mixture, and DNA from atransgenic founder mouse was shown.

FIG. 6. Spleen tissue of DKO-hu HSC mice at 20× and 40×, Mock versusvaccinated with HBsAg (the surface antigen of HBV).

FIG. 7. ELISPOT assay of splenocytes from mock or immunized DKO-hu miceshows that the HBV vaccine induced IgM+ B cells, but not IgG+ B cells.However, IgG+ B cell induction is achieved with anti-CD3/CD28 mAb (Tactivation) in vitro.

FIG. 8. Antigen-Fe fusion proteins give enhanced antigen-specific IgGinduction in vivo.

FIGS. 9A-9C. Dimer injection in AFC8/DKO mice leads to transient mouseliver damage and enhanced human hepatocyte engraftment (50-100×).AFC8/DKO transgenic mice were treated with AP20187 (5 μg/g). FIG. 9A.Plasma ALT levels were measured at 24 hr and 72 hr (and 3-6 days, notshown) post treatment. A second injection of AP20187 6 days after 1stinjection again leads to elevated ALT levels. Two non-TG DKO mice wereused as controls (dashed line, background). FIG. 9B. Livers wereharvested at 24 h after the 2nd injection and H&E stained. Accumulationof fat droplets (arrows) was observed in TG (but not in Non-TG) livers.V, vein. FIG. 9C. Enhanced human albumin levels in AP20187-treatedAFC8-DKO-hu mice. AFC8/DKO or DKO mice were treated with AP20187 (5ug/g) one hour prior to intra-splenic injection of human hepatocytes(2×10e6/mouse). Sera were collected at 80 days post transfer and humanalbumin levels were determined by ELISA. Thus, one injection ofdimerizers significantly enhanced human hepatocyte engraftment (100×).

FIGS. 10A-10D. HBV infection of DKO-hu HSC/Hep mice led to elevatedliver inflammation and long term HBV infection. DKO-hu HSC/Hep mice wereinfected with HBV (patient serum, 1×10e9 HBV genomes/mouse) or mock. At37 wpi, two mock and two HBV-infected mice were terminated. FIG. 10A.HBV-infected DKO-hu HSC/Hep mice had enlarged livers with 2-3× morenucleated cells. Error bars indicate SD and p value is shown. FIG. 10B.H&E staining of mock and HBV-infected livers. Elevated infiltration ofleukocytes and vacuoles (arrows) were detected in the HBV-infectedliver. FIG. 10C. HBV genome DNA in the infected mouse liver. Liver DNA(5 ng) was used to amplify HBV genome (core sequences) by nested PCR(HBV core sequences). 1. mock liver DNA; 2. HBV-infected liver DNA; 3.3,000 HBV genomes+mock liver DNA; and 4. 300,000 HBV genomes+mock liverDNA. FIG. 10D. Detection of HBV+ cells with the Anti-HBV core antibodyin the HBV infected DKO-hu liver. Liver section from the infected mousewas stained with anti-HBV core antibody, and HBV+ cells are indicated bythe arrows. Isotype controls or mock liver sections show no signals (notshown).

FIGS. 11A-11C. HCV infection in DKO-hu HSC/Hep mice is associated withelevated levels of T cell activation and liver pathology. DKO-hu HSC/Hepmice were infected with HCV (patient serum, 1×10e7 HCV genomes/mouse) ormock. At 25 wpi, two mock and one HCV-infected mice were analyzed. FIG.11A. HCV genome RNA in the infected mouse blood was detected (2×10e5copies/ml). Mock infected mice showed background level and patient HCVstock was also used as a control. FIG. 11B. HCV-infected DKO-hu HSC/Hepmice had higher levels of activated CD4 or CD8 T cells (% HLA-DR+). FIG.11C. H&E and anti-human albumin staining of HCV-infected livers. Humanalbumin+ hepatocytes and vacuoles were detected in the HCV-infectedliver.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thedisclosures of all United States patent references cited herein are tobe incorporated by reference to the extent they are consistent with thedisclosure herein.

As used herein in the description of the invention and the appendedclaims, the singular forms “a,” “an” and “the” are intended to includethe plural forms as well, unless the context clearly indicatesotherwise. Furthermore, the terms “about” and “approximately” as usedherein when referring to a measurable value such as an amount of acompound, dose, time, temperature, and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount. Also, as used herein, “and/or” or “/” refers to and encompassesany and all possible combinations of one or more of the associatedlisted items, as well as the lack of combinations when interpreted inthe alternative (“or”).

An improved non-human animal model of hepatitis infection is providedherein. The model has well-studied hemato-lymphoid cells and livertarget cells which are human, and HCV can establish infection and leadto T cell tolerance with similar features of T cell responses as in HCVinfected human or chimps. In some embodiments, the non-human animal isgenetically inbred, inexpensive and can be manipulated by genetic,immunological and pharmacological means.

The model is also useful for studying HCV/HBV and HIV co-infection andfor identifying or confirming therapeutic strategies for controllinghepatitis diseases. The model is further useful for studying human liverdevelopment/regeneration, identifying and screening compounds, anddetermining the pharmacokinetics of preclinical drugs.

Besides infection and immuno-pathogenesis, the model is useful to studyhuman liver stem cells, hepatocyte development/liver regeneration andautoimmune hepatitis. First, different subsets of human hepatocyteprogenitors can be tested. Second, the non-human animal is efficientlyreconstituted with a functional human immune system with T, B andmyeloid cells in lymphoid organs including liver. These human immunecells are present as normal resting cells and respond to antigenicstimulation in vitro or in vivo (Gimeno et al. 2004. Monitoring theeffect of gene silencing by RNA interference in human CD34+ cellsinjected into newborn RAG2−/− gammac−/− mice: functional inactivation ofp53 in developing T cells. Blood 104:3886-93; Traggiai et al. 2004.Development of a human adaptive immune system in cord bloodcell-transplanted mice. Science 304:104-7; and FIG. 3). The model isfurther useful for human liver regeneration, immuno-pathogenesis andimmune-based therapeutics. With the model including a human liver, drugliver toxicity, metabolism, and pharmacokinetics may also be tested.

“DKO-hu HSC” as used herein refers to the RAG2−/− γC−/− double knockoutnon-human mammal (with the suicidal transgene) comprising a human immunesystem formed from human hematopoietic stem cell administration.

“DKO-hu HSC/Hep” as used herein refers to the DKO-hu HSC non-humanmammal (with the suicidal transgene) further comprising human livercells (e.g., human hepatocytes). “Hepatitis” is an inflammation of theliver characterized by the presence of inflammatory cells in the liver.Acute hepatitis lasts for less than 6 months, while chronic hepatitislasts 6 months or longer. Causes of hepatitis include, but are notlimited to, certain viruses, toxins (e.g., alcohol), infectionselsewhere in the body, an autoimmune response, metabolic disease, etc.Hepatitis caused by viruses include, but are not limited to, HepatitisA, Hepatitis B, Hepatitis C, Hepatitis D (as a co-infection withHepatitis B virus), Hepatitis E, Hepatitis F, Hepatitis G, Herpessimplex, Cytomegalovirus, Epstein-Barr, yellow fever, adenovirus, etc.

“Hepatitis C virus” or “HCV” is the virus that causes Hepatitis C. AcuteHepatitis C symptoms, if present, are mild, and therefore the infectionis often not diagnosed at this early stage. About 20-30% of peopleinfected with HCV will clear the virus from their systems during theacute phase (i.e., the first 6 months after infection). The remaining70-80% will develop a chronic infection (i.e., lasting 6 months orlonger). Symptoms during the chronic phase are normally also mild, untilsignificant scarring of the liver has occurred. Once chronic, there islittle chance of clearing the virus without medical treatment.Co-infection of Hepatitis C with HIV, which is also a blood-borne virus,is common in the United States, particularly among intravenous drugusers.

“Hepatitis B virus” or “HBV” is the virus that causes Hepatitis B.Children are more susceptible than adults to develop a chronic HBVinfection. While more than 95% of adults are able to clear the viruswithout medical treatment, 70% of children ages 1-6, and only 5% ofnewborns who acquire HBV from their mothers can clear the infectionwithout treatment. Those who do not clear the virus become chroniccarriers.

“HIV” is the human immunodeficiency virus. It is a retrovirus that canlead to acquired immune deficiency syndrome (AIDS), a condition in whichthe immune system is compromised and the body is susceptible toopportunistic diseases that can be life-threatening. HIV primarilyinfects immune system cells, and leads to a decrease in the number ofCD4+ T cells, which are important for cell-mediated immunity. The mostcommon strain of HIV is HIV-1. A second strain, HIV-2, is also known.

“Transplanting,” “engrafting” or “grafting” is the placement of cells ortissue originating from one animal (e.g., a human) into another (e.g., amouse). In some embodiments cells are autogeneic (i.e., from the sameindividual animal or subject), isogeneic (i.e., a genetically identicalbut different animal or subject, e.g., from an identical twin, alsoknown as syngeneic), allogeneic (i.e., from a non-genetically identicalmember of the same species) or xenogeneic (i.e., from a member of adifferent species, also known as xenografting). In some embodiments,stem/progenitor cells are engrafted into the non-human animal.

“Infectivity” is the characteristic of an infectious agent (i.e., anagent that causes infection) that enables it to enter, survive andmultiply in a suitable host. Similarly, an “infection” is the invasionby and multiplication of one or more pathogenic microorganisms (e.g.,viruses, bacteria, etc.) in a bodily part or tissue, which may producesubsequent tissue injury and progress to overt disease, or thepathological state resulting from having been infected by suchmicroorganisms.

“Immunopathogenesis” includes the cellular and mechanistic eventsunderlying the typical course of development of an infection or diseasethat involves the immune response or the products of an immune response.In some embodiments, immunopathogenesis includes a dysfunction of theimmune response (e.g., HIV infection lowers T cell count).

A “human immune system phenotype” is an immune system of a non-humananimal that includes all or essentially all human cells of thehematopoietic lineages, including adaptive immune system cells such as Tcells, B cells, dendritic cells, natural killer (NK) cells, monocytesand macrophages, etc.

“Human” cells include, but are not limited to, cells that are directlyisolated from human tissue as well as cells derived from human cells,e.g., stem cells (such as hematopoietic stem cells) differentiated intoimmune system cells (e.g., in a non-human host animal, in vitro, etc.).

“Hematopoietic stem cells” are stem cells typically found in bonemarrow, cord blood, fetal liver and/or mobilized peripheral blood thatcan differentiate into all types of blood cells, including myeloid andlymphoid lineages. In some embodiments, hematopoietic stem cells aredirectly delivered (e.g., by direct injection) into the liver of theanimal (e.g., newborn mice).

The “adaptive immune system” includes immune cells that are able toremember and recognize antigens or pathogens that had previously evokedan immune response, and are thereby able to mount a stronger responseupon subsequent exposures, giving rise to “immunity” to a pathogen.

A “lymphocyte” is a type of white blood cell typically found invertebrates. Large granular lymphocytes include the natural killer (NK)cells, which are involved in innate immunity. Small lymphocytes includethe T cells and B cells, which are involved in adaptive immunity. Tcells (e.g., helper T cells, cytotoxic T cells) are typically involvedin the cell-mediated immune response, while B cells are typicallyinvolved in the humoral immune response. T cells and B cells typicallyrecognize non-self antigens presented on the surface of cells and elicitan immune response tailored to the non-self antigens. After activation,T cells and B cells typically leave behind memory cells that can elicita stronger response if the antigens are again detected.

A “human liver phenotype” is a liver organ of a non-human animal thatincludes human liver cells. “Human liver cells” are cells that normallyform the liver organ in humans, and include, but are not limited to,human hepatoblasts, hepatocytes, hepatic stellate cells, Kupffer cells,sinusoidal endothelial cells, lymphoid cells, etc.

“Hepatocytes” are the primary cells of the liver organ. Human livercells may also include stem/progenitor cells of the liver (e.g.,hepatocyte progenitor cell). “Hepatoblasts” are fetal liverstem-progenitor cells.

In some embodiments, human liver cells (e.g., hepatoblasts) are directlydelivered (e.g., by direct injection) into the liver of the animal(e.g., newborn mice). Other methods for the introduction of human livercells into a non-human animal are known in the art. See, e.g., U.S. Pat.No. 7,161,057 to Kneteman et al., which discusses infusing hepatocytesinto the spleen of the animal.

In some embodiments, the liver organ of the non-human animal comprisesat least 10, 20, 30, 40, or 50% of one or more types of humanliver/leukocyte cells (e.g., hepatocytes) by weight, by volume and/or bycell count. In some embodiments, the liver organ comprises at least 60,70 or 80% of human liver/leukocyte cells by weight, by volume or by cellcount.

“Isolated” signifies that the cells are placed into conditions otherthan their natural environment. However, the term “isolated” does notpreclude the later use of these cells thereafter in combinations ormixtures with other cells.

“Non-human animals” of the present invention are, in general, mammalsincluding primates, such as monkeys, more preferably rodents, and aremore particularly mice and rats. Animals may be male or female, and maybe of any age including adult. In some embodiments animals arelaboratory animals (e.g., non-human primates, rodents, dogs, pigs,birds, etc.). In some embodiments animals are mammalian laboratoryanimals.

A “recombinant” or “transgenic” non-human animal refers to a non-humananimal that has a genome or genetic material that is augmented oraltered in some fashion with a construct comprising a recombinantnucleic acid (e.g., a “transgene”) that is introduced into one or moreof the somatic and/or germ cells of the mammal. The nucleic acid orportions thereof may be, for example, of the same species (homologous)or of another species (heterologous) with respect to the host mammal.

A “recombinant” nucleic acid refers to a nucleic acid that has beenmanipulated in vitro, for example, by molecular biology techniques asdescribed herein and as known in the art.

A “knockout” of a target gene refers to an alteration in a host cellgenome that results in altered expression of the target gene (typicallya reduction in expression), e.g., by introduction of a mutation into acoding or noncoding region of the target gene, which mutation altersexpression of the target gene. Mammals may be heterozygous or homozygouswith respect to the mutation or insertion that causes the knockout.

“Wild type” gene or protein sequences of a given species are those DNAor protein sequences that are generally accepted in the art as being themost highly conserved within or across species.

By the term “express” or “expression” of a nucleic acid coding sequence,it is meant that the sequence is transcribed, and optionally,translated. Transcription can be measured by any means well known bythose of skill in the art, e.g., measuring the relative levels of mRNAexpression (e.g., with a northern blot, quantitative PCR, etc.).Typically, expression of a coding region will result in production ofthe encoded protein or polypeptide (measured by, e.g., western blot).

The production of transgenic animals is known and can be carried out inaccordance with known techniques or variations thereof which will beapparent to those skilled in the art, for example, as disclosed in: U.S.Pat. No. 7,022,893 to Takeda et al. and U.S. Pat. No. 6,218,595 to Giroset al., as well as U.S. Pat. No. 6,344,596 to W. Velander et al.(American Red Cross); U.S. Pat. No. 6,339,183 to T. T. Sun (New YorkUniversity); U.S. Pat. No. 6,331,658 to D. Cooper and E. Koren; U.S.Pat. No. 6,255,554 to H. Lubon et al. (American National Red Cross;Virginia Polytechnic Institute); U.S. Pat. No. 6,204,431 to P. Prieto etal. (Abbott Laboratories); U.S. Pat. No. 6,166,288 to L. Diamond et al.(Nextran Inc., Princeton, N.J.); U.S. Pat. No. 5,959,171 to J. M.Hyttinin et al. (Pharming BV); U.S. Pat. No. 5,880,327 to H. Lubon etal. (American Red Cross); U.S. Pat. No. 5,639,457 to G. Brem; U.S. Pat.No. 5,639,940 to I. Garner et al. (Pharmaceutical Proteins Ltd.;Zymogenetics Inc); U.S. Pat. No. 5,589,604 to W. Drohan et al. (AmericanRed Cross); U.S. Pat. No. 5,602,306 to Townes et al. (UAB ResearchFoundation); U.S. Pat. No. 4,736,866 to Leder and Stewart (Harvard); andU.S. Pat. No. 4,873,316 to Meade and Lonberg (Biogen).

Human immune system. Preferably, the non-human animal has an immunesystem comprising human immune cells. The transplantation of human CD34+cells into SCID or NOD/SCID mice leads to the generation of mainly humanmyeloid and B cells in the mouse bone marrow, but inefficient peripheralengraftment of human cells, especially human T cells (Lapidot et al.1992. Cytokine stimulation of multilineage hematopoiesis from immaturehuman cells engrafted in SCID mice. Science 255:1137-41; Larochelle etal. 1996. Identification of primitive human hematopoietic cells capableof repopulating NQD/SCID mouse bone marrow: implications for genetherapy. Nat Med 2:1329-37). More recently, a mouse model with afunctional human immune system has been reported. The Rag2-γC doubleknockout (DKO) mouse lacks T, B and NK cells, and serves as better hostsfor engraftment of human cells/tissues. Therefore, in preferredembodiments the non-human animal is a Rag2-γC double knockout (DKO)transgenic non-human animal.

In some embodiments, cord blood CD34+ human HSC are injected directlyinto the liver of newborn DKO animals (see Traggiai et al. 2004.Development of a human adaptive immune system in cord bloodcell-transplanted mice. Science 304:104-7). The new born liverenvironment appears to support efficient human HSC engraftment andreconstitution of the animal with a functional human immune system incentral and peripheral lymphoid organs.

Remarkably, long term human T cell development occurs efficiently in themouse DKO thymus, and normal human T, B, NK and dendritic cells arereadily detected in peripheral lymphoid tissues such as spleen, lymphnodes (LN) and peripheral blood (PB). Importantly, de novo human B and Tcell responses are elicited in the hu-HSC-DKO mouse by standardimmunization (human TT-specific IgG induction) or infection with thehuman tumor virus EBV (expansion of EBV-specific CD8 T cells).

Both CCR5 and CXCR4 are expressed on human immature and mature T cells(Zhang et al. 2007. HIV-1 infection and pathogenesis in a novelhumanized mouse model. Blood 109:2978-81). DKO-hu HSC mice allowefficient HIV-1 infection with high plasma viremia. High levels ofproductive infection occur in the thymus, spleen and lymph nodes. HumanCD4+ T cells are gradually depleted by HIV-1 in a dose-dependent manner.In addition, HIV-1 infection persists in infected DKO-hu HSC mice for atleast 19 weeks, with infectious HIV-1 in lymphoid tissues. Thus, theDKO-hu HSC mouse can serve as a relevant in vivo model to investigatemechanisms of HIV-1 infection and immuno-pathogenesis.

Combined with engrafted human liver cells, the HSC-DKO mouse asdescribed herein can serve as a model to investigate mechanisms of HCVimmuno-pathogenesis and how coinfection with HIV-1 may affect HCVreplication and/or pathogenesis.

Human liver cells. In preferred embodiments, the non-human mammalcomprises human liver cells. Human liver cells may be introduced intothe non-human mammal by the procedures provided herein or by proceduresknown in the art (see, e.g., U.S. Pat. No. 7,161,057 to Kneteman,incorporated by reference herein).

In some embodiments, human hepatoblasts/progenitors are isolated fromhuman fetal liver tissues. Hepatocytes (or parenchyma cells) may beisolated from livers by collagenase digestion as described (Meuleman etal. 2005. Morphological and biochemical characterization of a humanliver in a uPA-SCID mouse chimera. Hepatology 41:847-56; Schmelzer etal. 2006. The phenotypes of pluripotent human hepatic progenitors. StemCells 24:1852-8). EpCAM, a transmembrane glycoprotein, has been shown tomark human hepatic stem or progenitor cells as it is expressed byhepatic progenitors but not hepatocytes (de Boer et al. 1999. Expressionof Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. JPathol 188:201-6). Transplantation of EpCAM+ cells into the liver ofmice gives rise to human liver tissue expressing human-liver specificproteins (de Boer et al. 1999. Expression of Ep-CAM in normal,regenerating, metaplastic, and neoplastic liver. J Pathol 188:201-6;Schmelzer et al. 2006. The phenotypes of pluripotent human hepaticprogenitors. Stem Cells 24:1852-8).

In some embodiments, approximately one million (10⁶) CD34+ HSC cells areco-transferred with approximately one million (10⁶) parenchyma cells(comprising human hepatoblasts and/or hepatic stem cells) (105 EpCAM+hepatoblasts) into the liver of 1- to 3-day-old DKO or AFK8/DKO micepreviously irradiated at 400 rad (sublethal). In some embodiments, cellsare co-injected in the liver of newborn DKO mice.

Antagonistic antibody against c-met. To improve human hepatocyte growth,in some embodiments an agonistic antibody against human c-Met (c-MetmAb, mouse IgG1) is used that activates human but not murine c-Met asreported (Ohashi et al. 2000. Sustained survival of human hepatocytes inmice: A model for in vivo infection with human hepatitis B and hepatitisdelta viruses. Nat Med 6:327-31).

DKO-hu HSC/Hep mice in AFC8/DKO mice. In another embodiment, DKO-huHSC/Hep mice are generated in an AFC8/DKO background (FKBP-Casp8 geneunder control of the albumin promoter, see Heckel et al. 1990. Neonatalbleeding in transgenic mice expressing urokinase-type plasminogenactivator. Cell 62:447-56) (FIG. 6&7). Transfer of adult hepatocytesinto uPA-SCID mice has led to efficient engraftment of human hepatocytes(70%) in the chimeric liver (Mercer et al. 2001. Hepatitis C virusreplication in mice with chimeric human livers. Nat Med 7:927-33;Meuleman et al. 2005. Morphological and biochemical characterization ofa human liver in a uPA-SCID mouse chimera. Hepatology 41:847-56).However, the uPA-SCID or uPA-TKO mouse (Azuma et al. 2007. Robustexpansion of human hepatocytes in Fah(−/−)/Rag2(−/−)/I12rg(−/−) mice.Nat Biotechnol 25:903-10) has no immune system, is difficult to breedand not optimal for most studies. Hepatocytes of the AFC8/DKO mouse canbe inducibly depleted with the FKBP dimerizer ligand AP20187 (Burnett etal. 2004. Conditional macrophage ablation in transgenic mice expressinga Fas-based suicide gene. J Leukoc Biol 75:612-23; Pajvani et al. 2005.Fat apoptosis through targeted activation of caspase 8: a new mousemodel of inducible and reversible lipoatrophy. Nat Med 11:797-803).

Methods of screening compounds. The present invention also providesmethods of screening a compound for activity in treating hepatitisand/or HIV infection. In some embodiments the method comprisesadministering a test compound to a mammal as described herein, and thendetecting the presence or absence of activity in treating and/orpreventing hepatitis and/or HIV infection in the mammal. Theadministering step may be carried out by any suitable techniquedepending upon the particular compound, including parenteral injection,oral administration, inhalation administration, transdermaladministration, etc.

“Treat” refers to any type of treatment that imparts a benefit to asubject, e.g., a subject afflicted with or at risk for developing adisease or condition (e.g., a liver infection and/or HIV/AIDS, etc.).Treating includes actions taken and actions refrained from being takenfor the purpose of improving the condition of the subject (e.g., therelief of one or more symptoms), delay in the onset or progression ofthe disease, disease prevention (e.g., immunization), etc.

Production of monoclonal antibodies. Monoclonal antibodies can beproduced in a hybridoma cell line formed according to well-knowntechnique of Kohler and Milstein, (1975) Nature 265:495-97, using ahuman immune cell isolated from the spleen of a non-human mammal with ahuman immune system/human liver phenotype for the fusion. For example,human spleen cells are isolated from a non-human animal having a humanimmune system. The spleen cells are then immortalized by fusing themwith myeloma cells or with lymphoma cells, typically in the presence ofpolyethylene glycol, to produce hybridoma cells. The hybridoma cells arethen grown in a suitable medium and the supernatant screened formonoclonal antibodies having the desired specificity. Monoclonal Fabfragments can be produced in bacterial cells such as E. coli byrecombinant techniques known to those skilled in the art. See, e.g., W.Huse, (1989) Science 246:1275-81.

In some embodiments of the present invention, vaccination of the DKO-huHSC with a human HBV vaccine induces IgM-producing human B cells, butvery low levels of human IgG+ B cells. Human IgG+ B cells can beincreased by activating human T cells in the spleen in vitro e.g., withanti-CD3/CD28 mAb. Further, immunization of the non-human animal with aspecific protein may induce antigen-specific human IgG producing Bcells. In some embodiments, antigen-specific human IgG induction can beenhanced in vivo by using the fusion protein with the antigen and the Fcdomain of human IgG.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLE 1

Stable reconstitution of DKO mice with CD34+ HSC (DKO-hu HSC mice>1 yr).Human fetal liver derived CD34+ HSC (0.5-1×10⁶/mouse) were transplantedinto newborn DKO mice intra-hepatically. Functional lymphoid organs areformed in the DKO-HSC mice as reported (see Baenziger et al. 2006.Disseminated and sustained HIV infection in CD34+ cord bloodcell-transplanted Rag2−/−gamma c−/− mice. Proc Natl Acad Sci U.S.A.103:15951-6; Traggiai et al. 2004. Development of a human adaptiveimmune system in cord blood cell-transplanted mice. Science 304:104-7;Zhang et al. 2007. HIV-1 infection and pathogenesis in a novel humanizedmouse model. Blood 109:2978-81). Experiments showed that >95% of thetransplanted DKO mice have stable human cell engraftment with humanCD45+ cells in the blood for at least 50 weeks (FIG. 1A; Zhang et al.2007. HIV-1 infection and pathogenesis in a novel humanized mouse model.Blood 109:2978-81; and data not shown). T, B, monocytes, mDC and PDC arestably reconstituted (FIG. 1B and data not shown). About 30×10⁶ totalsplenocytes (40% of wild type mice) and 20×10⁶ thymocytes (20% of wildtype mice) are generated in DKO-hu mice (FIG. 1C). Most CD4 and CD8 Tcells express a resting naïve phenotype (CD45RO-CD69− and CD62L+CCR7+,FIG. 1D and data not shown). When their proliferation response to TCRstimulation is compared to human CD4 T cells (FIG. 1F), DKO-hu derivedhuman CD4 T cells (FIG. 1E) show identical response to different dosesof anti-CD3 mAb and to CD28 co-stimulation.

Human T cells are negatively selected by both mouse MHC/APC and humanMHC/APC because both human and mouse antigen presenting cells (APC) aredetected in the mouse thymus (see Traggiai et al. 2004. Development of ahuman adaptive immune system in cord blood cell-transplanted mice.Science 304:104-7). Thus, human T cells developed in the DKO-hu HSCmouse are tolerized to both BalbC mouse cells and to human cells from“cohort-mate” DKO-hu mice of the same donor (“inbred/syngeneic” hu-mice)shown by lack of mixed lymphocyte response (MLR) between splenocytesfrom DKO, A1 and B DKO-hu mice, and between “syngeneic” A1 and A2 DKO-humice. However, either A1 or A2 cells react strongly with the“allogeneic” cells from the cohort B DKO-hu mice (FIG. 1G). In addition,immunization of DKO-hu mice with Ova protein induces Ova-specific humanT and B cell responses (FIG. 1H and data not shown). Therefore, normalhuman T and B cells are generated in the DKO-hu mouse.

EXAMPLE 2

HIV-1 infection and pathogenesis in DKO-hu HSC mice. Both CCR5 and CXCR4are expressed on human immature and mature T cells in DKO-hu mice.DKO-hu HSC mice allow efficient HIV-1 infection with high plasmaviremia. High levels of productive infection occur in the thymus, spleenand lymph nodes. Interestingly, both CD45RO+ (memory/effector) andCD45RO− (naïve) CD4T cells are productively infected as stained for HIVgag p24. Human CD4+ T cells are rapidly depleted by a pathogenicHIV-1-R3A (FIG. 2A/B). In addition, HIV-1 infection persists in infectedDKO-hu HSC mice for at least 19 weeks, with infectious HIV-1 in lymphoidtissues. DKO-hu mice were also infected with the less pathogenic HIV-1isolate JRCSF (CCR5-tropic). High levels of HIV replication weredetected at 1 to 18 weeks post infection (FIG. 2C). CD4+ T cells in theblood were only slowly decreased but maintained at steady levels for13-128 weeks (FIG. 2D). When lymphoid organs were analyzed from R3A-(2wpi) or JRCSF-(4 wpi) samples, R3A infection almost completely depletedhuman CD4+ T cells in the spleen and dramatically depleted CD4 T cellsin mLN, whereas JRCSF infection did not significantly deplete human CD4T cells in the spleen or mLN (FIG. 2E-H). Interestingly, there is anincrease in the CD8 levels in the mLN for both infections.

At 22 wpi with JRCSF, HIV infection is associated with an enlargedspleen and activated HLA-DR+ T cells in DKO-hu mice. CD4 depletion isalso observed in lymph node organs and the remaining T cells showactivated phenotypes. Therefore, HIV infection leads toimmuno-pathogenesis, correlated with hyper-immune activation andinflammation. Indeed, inflammatory cytokines are induced by HIVinfection in plasma as well as in lymphoid tissues (data not shown).

Thus, the DKO-hu HSC mouse can serve as a relevant in vivo model toinvestigate mechanisms of HIV-1 infection and immuno-pathogenesis.HIV-R3A can be used to study acute HIV infection with a rapid CD4depletion, and JRCSF can be used to study acute HIV infection,immuno-response and chronic HIV infection and immunopathogenesis.

EXAMPLE 3

Development of the DKO-hu HSC/Hep mouse. To develop the DKO-hu HSC/Hepmice, we isolated human CD34+ HSC and human hepatoblasts/progenitorsfrom human fetal liver tissues. Hepatocytes (or parenchyma cells) areisolated from livers by collagenase digestion as described (Meuleman etal. 2005. Morphological and biochemical characterization of a humanliver in a uPA-SCID mouse chimera. Hepatology 41:847-56; Schmelzer etal. 2006. The phenotypes of pluripotent human hepatic progenitors. StemCells 24:1852-8). EpCAM, a transmembrane glycoprotein, has been shown tomark human hepatic stem or progenitor cells as it is expressed byhepatic progenitors but not hepatocytes (de Boer et al. 1999. Expressionof Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. JPathol 188:201-6). Transplantation of EpCAM+ cells into the liver ofmice gave rise to human liver tissue expressing human-liver specificproteins (de Boer et al. 1999. Expression of Ep-CAM in normal,regenerating, metaplastic, and neoplastic liver. J Pathol 188:201-6;Schmelzer et al. 2006. The phenotypes of pluripotent human hepaticprogenitors. Stem Cells 24:1852-8).

One million (1×10e6) CD34+ HSC cells are co-transferred with 1×10e6parenchyma cells (10e5 EpCAM+ hepatoblasts) into the liver of 1- to3-day-old DKO or AFK8/DKO mice previously irradiated at 400 rad(sublethal). 20-30 DKO-hu mice are generated from each fetal livertissue donor.

Human T/B/DC cells are analyzed by FACS at different time points afterHSC transplant as previously reported (FIG. 1A-1H; Meissner et al. 2004.Characterization of a thymus-tropic HIV-1 isolate from a rapidprogressor: role of the envelope. Virology 328:74-88; Su et al. 1995.HIV-1-induced thymocyte depletion is associated with indirectcytopathogenicity and infection of progenitor cells in vivo. Immunity2:25-36). Human cells (CD45+) are analyzed for CD4, CD8, CD25 (Treg),HLA-DR (activated T cells), CD27/CD45RO (naïve and memory T cellsubsets), CD19 (B cells), CD11c (mDC) and CD123 (PDC). Antibodies withappropriate labels are purchased from BD-Pharmingen.

When the parenchyma cell suspensions prepared from fetal livers wereanalyzed, 12% of liver cell suspensions from fetal livers are EpCAM+cells, of which more than 80% are hepatoblasts and hepatic stem cells(Schmelzer et al. 2006. The phenotypes of pluripotent human hepaticprogenitors. Stem Cells 24:1852-8). Thus, the parenchyma cells preparedfrom fetal livers are enriched with human hepatoblasts.

These cells are co-injected in the liver of newborn DKO mice. As shownabove, human CD34+ HSC reconstituted the human blood system with afunctional human immune system. Regarding the hepatocyte reconstitution,human liver stem/progenitor cells have been documented in both CD34+cells (Dan et al. 2006. Isolation of multipotent progenitor cells fromhuman fetal liver capable of differentiating into liver and mesenchymallineages. Proc Natl Acad Sci USA 103:9912-7) and in hepatocyte-likeparenchyma EpCAM+ cells (Schmelzer et al. 2006. The phenotypes ofpluripotent human hepatic progenitors. Stem Cells 24:1852-8). Theseprogenitor cells injected in the liver give rise to human hepatocytes inthe chimeric mouse.

When CD34+ cells were co-transplanted with parenchyma hepatoblasts,significant and stable human albumin in the chimeric mouse blood wasdetected (FIG. 3A-3F). The engraftment of human blood cells wasmonitored by FACS for human leukocytes (>20% in total PBMC). Humanalbumin in the plasma of mice was measured (100-500 ng/ml) by ELISAassay (FIG. 3F) and, for human hepatocytes, by IF staining of human Alb+cells in the liver (FIG. 3C-E).

EXAMPLE 4

Multiple types of human cells are developed in the liver of DKO-huHSC/Hep mice. When liver leukocytes from the DKO-hu HSC mouse wereisolated, 80% showed human CD45+ expression. Both lymphoid cells andmyeloid cells were present in the reconstituted liver (FIG. 3A-B).Immuno-staining of liver section also demonstrates reconstitution ofhuman hepatocytes (Albumin+, FIG. 3C-E). Therefore, human hepatocytes aswell as human T/B/myeloid cells are present in the chimeric liver.

EXAMPLE 5

FKBP-Casp8 transgenic mice with inducible death of liver cells. TheDKO-hu HSC/Hep mouse model was improved by 1) boosting human hepatocytecell growth with anti-human c-Met mAb, and 2) by selective ablation ofmurine hepatocytes (AFC8/DKO-hu HSC/Hep mice).

The newborn BalbC/Rag2^(−/−)γ_(c) ^(−/−) DKO mouse is currently the mostpermissive mouse model for the engraftment of human tissues, allowinglong-term development of human primary and secondary immune organs. Whenhuman liver stem/progenitor cells (hepatoblasts) are transplanted intonewborn (1-3 days) DKO mice, human liver cells are also detected in thechimeric mice. However, the levels of human hepatocyte engraftment isrelatively low. Therefore, to enhance human liver cell engraftment inthe DKO-hu mouse, murine hepatocyte death is induced after human livercell transfer.

The FKBP-Caspase8 fusion gene with inducible cell killing activity wasconstructed (FIG. 4A-4C; and Chang et al. 2003. Activation ofprocaspases by FK506 binding protein-mediated oligomerization. Sci STKE2003:PL1). Addition of FKBP dimerizer AP20187 to cells transfected withthe FKBP-Casp8 and/or GFP led to death of GFP+/FKBP-Casp8+ cells, butnot GFP− bystander cells, in a dose-dependent fashion (FIG. 4B/C). Thedrug had no detectable effect on cells transfected with only GFPexpressing gene. Therefore, FKBP-Casp8 activation by AP20187 killstarget cells expressing the gene but not bystander cells, an importantpoint for depleting specific target cells in vivo.

DKO female mice are super-ovulated and fertilized eggs are isolated bystandard procedures in the UNC transgenic core facility. TheAlb-FKBP-Casp8 transgene is injected into fertilized DKO eggs andimplanted into surrogate mother mice. Screening for the Alb-FKBP-Casp8transgene is done with PCR (FIG. 5C; Kovalev et al. 2001. An importantrole of CDK inhibitor p18(INK4c) in modulating antigen receptor-mediatedT cell proliferation. J Immunol 167:3285-92). FKBP-Casp8/DKO transgenicfounders are confirmed by Southern blot and its expression confirmed bywestern/IF (anti-human Caspase 8 mAb) of liver tissues. Spleen, kidney,heart and thymus are used as control tissues. After establishing theAFC8/DKO founder mice that express the fusion protein in the liver, wetest the dose and duration of AP20187 to induce hepatocyte apoptosis inthe mouse. For peritoneal injections, AP20187 (Ariad Pharmaceuticals) isprepared (1 mg/ml in a solution consisting of 4% ethanol, 10% PEG-400,and 1.7% Tween). The AP20187 dose is adjusted to deliver 0.25, 1, 4 and10 mg/kg (3-5 mice/dose). This dose range of AP20187 is effective ininducing Capsase8 activation and target cell depletion. No toxicity isobserved in mice because AP20187 is engineered for in vivo purposes anddoes not interact with endogenous FKBP (Burnett et al. 2004. Conditionalmacrophage ablation in transgenic mice expressing a Fas-based suicidegene. J Leukoc Biol 75:612-23; Pajvani et al. 2005. Fat apoptosisthrough targeted activation of caspase 8: a new mouse model of inducibleand reversible lipoatrophy. Nat Med 11:797-803).

The AFC8/DKO mouse was used to construct AFC8/DKO-hu HSC/Hep mice asdescribed above. At 3-8 weeks post transplant of human cells, AP20187 isadministered to induce death of murine hepatocytes. Hepatocyte depletionis monitored by measuring serum ALT level and increased humanhepatocytes by blood levels of human albumin at 1, 2, 4, 6 and 8 weekspost drug treatment. At 2, 4 and 8 weeks post induction, we harvest thechimeric liver of selected AFC8/DKO-hu HSC/Hep mice to monitor murinehepatocyte death (apoptosis markers) and human hepatocytes (humanalbumin+ cells), and expression of other human liver-specific geneslisted above.

For peritoneal injections, AP20187 (Ariad Pharmaceuticals) is prepared(1 mg/ml in a solution consisting of 4% ethanol, 10% PEG-400, and 1.7%Tween). The AP20187 dose is adjusted to deliver 0.25, 1, 4 and 10 mg/kg(3-5 mice/dose). This dose range of AP20187 is effective in inducingCapsase8 activation and target cell depletion. No toxicity is observedin mice because AP20187 is engineered for in vivo purposes and does notinteract with endogenous FKBP (Burnett et al. 2004. Conditionalmacrophage ablation in transgenic mice expressing a Fas-based suicidegene. J Leukoc Biol 75:612-23; Pajvani et al. 2005. Fat apoptosisthrough targeted activation of caspase 8: a new mouse model of inducibleand reversible lipoatrophy. Nat Med 11:797-803).

When expressed from the liver-specific albumin promoter in thetransgenic construct (FIG. 5A; and Heckel et al. 1990. Neonatal bleedingin transgenic mice expressing urokinase-type plasminogen activator. Cell62:447-56), FKBP-Casp8 only kills HepG2 cells, but not 293T cells, in adimer-dependent fashion (FIG. 5B). This confirms the hepatocyte-specificexpression of the transgene. DKO transgenic mice have been generatedwith the Alb-FKBP-Casp8 construct (FIG. 5C).

Human hepatocyte level and functions are closely monitored by measuringhuman albumin expression. Expression of human liver-specific markergenes including human albumin, α-fetoprotein (AFP, highly expressed infetal liver but not adult liver), CytochromeP450/CYP3A4 and CYP1A2 aremeasured by TaqMan RT-PCR and by detecting protein expression by IF, asreported (Azuma et al. 2007. Robust expansion of human hepatocytes inFah(−/−)/Rag2(−/−)/I12rg(−/−) mice. Nat Biotechnol 25:903-10; Meulemanet al. 2005. Morphological and biochemical characterization of a humanliver in a uPA-SCID mouse chimera. Hepatology 41:847-56).

Dimer injection in AFC8/DKO mice led to transient mouse liver damage andenhanced human hepatocyte engraftment (50-100×) (FIG. 9A-9C). AFC8/DKOtransgenic mice were treated with AP20187 (5 μg/g). Plasma ALT levelswere measured at 24 hr and 72 hr (and 3-6 days, not shown) posttreatment (FIG. 9A). A second injection of AP20187 6 days after 1stinjection again led to elevated ALT levels. Two non-TG DKO mice wereused as controls (dashed line, background). Livers were harvested at 24h after the 2nd injection and H&E stained (FIG. 9B). Accumulation of fatdroplets (arrows) was observed in TG (but not in Non-TG) livers. V,vein. Enhanced human albumin levels were found in AP20187-treatedAFC8-DKO-hu mice (FIG. 9C). AFC8/DKO or DKO mice were treated withAP20187 (5 μg/g) one hour prior to intra-splenic injection of humanhepatocytes (2×10e6/mouse). Sera were collected at 80 days post transferand human albumin levels were determined by ELISA. Thus, one injectionof dimerizers significantly enhanced human hepatocyte engraftment(100×).

The improved DKO-hu HSC/Hep mouse is used for HBV, HCV and/or HIVinfection.

EXAMPLE 6

Agonistic antibody against human c-Met. Hepatocyte Growth Factor (HGF)binds to c-Met (the HGF receptor) and is essential in the developmentand regeneration of the liver. To improve human hepatocyte growth, anagonistic antibody against human c-Met is used (c-Met mAb, mouse IgG1)that activates human but not murine c-Met as reported (Ohashi et al.2000. Sustained survival of human hepatocytes in mice: A model for invivo infection with human hepatitis B and hepatitis delta viruses. NatMed 6:327-31).

In each cohort, 50% of the DKO-HSC/Hep mice are injected i.v. with theanti-C-Met mAb 3D1 (Genentech, South San Francisco, Calif.), at 50μg/mouse weekly from 2-8 weeks post transplant (Ohashi et al. 2000.Sustained survival of human hepatocytes in mice: A model for in vivoinfection with human hepatitis B and hepatitis delta viruses. Nat Med6:327-31). Mouse IgG1 is used for controls. Human albumin levels aremeasured weekly in blood before, during and after treatment, and humanhepatocytes are measured by IF of liver sections. Proliferation of humanhepatocytes (Alb+) is analyzed by Ki67 staining or by in vivo BrDU pulselabeling followed by FACS or IF (Kovalev et al. 2001. An important roleof CDK inhibitor p18(INK4c) in modulating antigen receptor-mediated Tcell proliferation. J Immunol 167:3285-92). 3-4 cohorts are tested tomonitor the anti-c-Met effect on human hepatocyte engraftment.

EXAMPLE 7

Production of Antigen-specific Human IgG in DKO-hu HSC Mice. Vaccinationof the DKO-hu HSC mouse with a human HBV vaccine induced onlyIgM-producing human B cells but very low levels of human IgG+ B cells(FIG. 6). Human IgG+ B cells were increased by activating human T cellsin the spleen in vitro with anti-CD3/CD28 mAb (FIG. 7). Similarly,immunization with the chicken ovalbumin protein induced very low levelsof Ova-specific human IgG (FIG. 8). Ova-specific human IgG induction isenhanced by using the fusion protein between the same chicken ovalbuminprotein with the Fc domain of human IgG (FIG. 8).

EXAMPLE 8

HBV infection of DKO-hu HSC/Hep mice leads to elevated liverinflammation and long term HBV infection. DKO-hu HSC/Hep mice wereinfected with HBV (patient serum, 1×10e9 HBV genomes/mouse) or mock. At37 wpi, two mock and two HBV-infected mice were terminated.

HBV-infected DKO-hu HSC/Hep mice had enlarged livers with 2-3× morenucleated cells (FIG. 10A), and elevated infiltration of leukocytes andvacuoles (arrows) were detected in the HBV-infected liver (FIG. 10B).The HBV genome DNA was detected in the infected mouse liver (FIG. 10C),and HBV+ cells were also detected with the Anti-HBV core antibody (FIG.10D).

EXAMPLE 9

HCV infection in DKO-hu HSC/Hep mice is associated with elevated levelsof T cell activation and liver pathology. DKO-hu HSC/Hep mice wereinfected with HCV (patient serum, 1×10e7 HCV genomes/mouse) or mock. At25 wpi, two mock and one HCV-infected mice were analyzed. A. HCV genomeRNA in the infected mouse blood was detected (2×10e5 copies/ml). Mockinfected mice showed background level and patient HCV stock was alsoused as a control. B. HCV-infected DKO-hu HSC/Hep mice had higher levelsof activated CD4 or CD8 T cells (% HLA-DR+). C. H&E and anti-humanalbumin staining of HCV-infected livers. Human albumin+ hepatocytes andvacuoles were detected in the HCV-infected liver. HCV+ hepatocytes inthe liver tissue are also measured.

HCV genomes were detected in the infected DKO-hu mice at 25 wpi, but notin mock infected DKO-hu mice or HCV-infected DKO mice (FIG. 11A and datanot shown). In addition, elevated levels of activated human T cells weredetected (FIG. 11B), as well as liver pathology characteristic ofvirus-induced hepatitis/liver diseases (FIG. 11C).

That which is claimed is:
 1. A recombinant non-human mammal comprising:(a) an immune system comprising: human T cells, human B cells, humannatural killer cells, human monocytes and macrophages and humandendritic cells, so that said mammal expresses a human immune systemphenotype; and (b) a liver comprising human hepatocytes, so that saidmammal expresses a human liver phenotype.
 2. The mammal of claim 1,wherein said human liver cells comprise at least 20% by volume of saidliver of said mammal.
 3. The mammal of claim 1, wherein said mammal is aRag2-gammaC double knockout mammal.
 4. The mammal of claim 1, whereinsaid mammal comprises non-human cells that contain a liver-specificinducible promoter operatively associated with a nucleic acid encoding aproduct toxic to said non-human cells.
 5. The mammal of claim 4, whereinsaid liver-specific inducible promoter comprises a FKBP induciblepromoter and said nucleic acid encoding a product toxic to saidnon-human cells comprises a Caspase8 gene.
 6. The mammal of claim 1,wherein said mammal is infected with a virus.
 7. The mammal of claim 1,wherein said mammal is infected with HIV-1 virus, a hepatitis virus, orboth.
 8. The mammal of claim 7, wherein said hepatitis virus isHepatitis B virus (HBV) or Hepatitis C virus (HCV).
 9. The mammal ofclaim 1, wherein said mammal is a mouse.
 10. A method of screening acompound for activity in treating hepatitis, comprising: administering atest compound to the recombinant non-human mammal of claim 1; and thendetecting the presence or absence of said activity in said mammal, saidpresence of said activity in said mammal indicating that said compoundhas activity in treating hepatitis.
 11. The method of claim 10, whereinsaid detecting step is carried out by a biochemical assay.
 12. A methodof making a non-human transgenic mammal comprising an immune system,said immune system comprising: human T cells, human B cells, humannatural killer cells, human monocytes and macrophages and humandendritic cells, so that said mammal expresses a human immune systemphenotype; and a liver comprising human hepatocytes, so that said mammalexpresses a human liver phenotype; said method comprising the steps of:(a) providing a BalbC/Rag2^(−/−)γ_(c) ^(−/−) double knockout transgenicmammal; (b) transplanting human CD34+ hematopoietic stem cells into saiddouble knockout transgenic mammal, wherein said stem cells differentiateinto human T cells, human B cells, human natural killer cells, humanmonocytes and macrophages and human dendritic cells in said transgenicmammal; and (c) transplanting human liver cells into said doubleknockout transgenic mammal, wherein said liver cells form humanhepatocytes in said liver of said transgenic mammal.
 13. The method ofclaim 12, wherein said human CD34+ hematopoietic stem cells and saidhuman liver cells are autogeneic with respect to each other.
 14. Themethod of claim 12, wherein said human liver cells comprise humanparenchyma hepatoblasts.
 15. The method of claim 12, wherein said humanCD34+ hematopoietic stem cells and said human liver cells aretransplanted simultaneously.
 16. The method of claim 12, wherein saidtransplanting steps are carried out when said transgenic mammal is from0 to 10 days old.
 17. The method of claim 12, wherein said transplantingsteps are carried out when said transgenic mammal is from 1-3 days old.18. The method of claim 12, wherein said transgenic mammal furthercomprises an Alb-FKBP-Casp8 transgene.
 19. The method of claim 12,further comprising the step of administering a c-Met agonist selectivefor human c-Met to said transgenic animal.
 20. The method of claim 19,wherein said c-Met agonist is an agonistic antibody against human c-Met.21. The method of claim 19, wherein said administering step is carriedout by injecting an anti-C-met antibody.
 22. A method of making fusioncells useful for the production of human monoclonal antibodies, saidmethod comprising: isolating a human antibody-secreting B lymphocytefrom a recombinant non-human mammal of claim 1; and then fusing saidantibody-secreting B lymphocyte with immortal cells to form said fusioncells.
 23. The method of claim 22, wherein said immortal cell is a humanor mouse myeloma cell.
 24. The method of claim 22, wherein saidantibody-secreting B lymphocyte is isolated from a spleen or lymph nodeof said recombinant non-human mammal.
 25. The method of claim 22,wherein said mammal is infected with a virus.
 26. The method of claim22, wherein said mammal is infected with HIV-1 virus, a hepatitis virus,or both.
 27. The method of claim 26, wherein said hepatitis virus isHepatitis B virus (HBV) or Hepatitis C virus (HCV).